Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein, factor VIII. There is no cure for hemophilia A and treatment consists of replacement using (purified) factor VIII preparations. During the last funding period we have made significant strides in characterizing sequences and ligands, and determining mechanisms important for factor VIIL/factor Villa subunit structure and function. We will use this information as a platform to identify fine point structural details of intra-protein interactions that will define mechanisms for the regulation of this critical protein. The first aim will study inter-factor Villa subunit interactions related to the retention and stimulation of A2 subunit. This subunit is critical as demonstrated by the limited cofactor activity observed for isolated A2. Further, the relatively weak affinity interaction of A2 subunit in factor VIIIa governs the subunit structure and function of the active protein. Goals are to identify and determine the roles of residues in A1 and A2 that serve in the inter-subunit interaction and are responsible for the stimulation of A2-dependent cofactor activity. Specific aims include: (i) elucidation of salt bridging between basic residues in A2 and the acidic C-terminus of Al and (ii) examination of interactive sites in Al that are independent of this C-terminal tail. Methods will employ peptide analysis and molecular biology/cell culture techniques to generate novel factor VIII/subunit molecules. Alterations in activity will be correlated with changes in structure using a variety of physical methods amenable to low levels of protein. The second aim studies metal ion-dependent and -independent interactions in factor VIII involved in association of heavy and light chains. The goal of this aim is to define and model the association of these two chains in the active conformation. Our studies will: (i) examine the role of Cu in the high affinity inter-chain interaction following characterization of factor VIH molecules possessing altered consensus type 1 and type 2 Cu binding Sites, (ii) determine the role of Ca2+ in yielding the active cofactor conformation following quantitation of Ca sites and physical analyses of the conversion of inactive to active cofactor conformation, and (iii) assess metal ion-independent interactions including the role of exposed hydrophobic sites in heavy and light chains, and contribution of the A2 domain to the inter-factor VIII interaction. Definition of these issues will yield valuable insights into the biochemistry of the native as well as dysfunctional factor VIII molecules, and provide information for the design of superior therapeutics.